Recombinant Expression of Fructosyl Transferase Gene of Aspergillus oryzae in Escherichia coli
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Abstract:
To obtain a higher yield of fructosyl transferase during industrial production, the fructosyl transferase gene from Aspergillus oryzae SBB201 strain was amplified using reverse transcriptase-polymerase chain reaction (RT-PCR) technique and cloned into plasmid pEASY-E1, which was then transformed into E. coli BL21-DE3 strain to achieve successful expression. Optimal conditions for inducing the expression of recombinant genes were ultimately determined for successful expression of fructosyl transferase, namely, induction temperature of 25 ℃ and a dose of 1.0 μmol/mL isopropylthiogalactoside (IPTG,) as an inducer. Taking advantage of the 6 × histidine-tag at the amino terminus of recombinant plasmid PEASY-S1, the expressed fructosyl transferase was purified by chelating affinity chromatography. Sucrose was used as substrate to test the catalyzing ability of fructosyl transferase. The results indicated that the recombinant fructosyl transferase had catalyzing ability and its enzyme activity reached 59.0 U/g. In addition, this recombinant fructosyl transferase could be stably expressed at low temperature. Thus, it has great potential for development and application. In this study, recombinant expression of fructosyl transferase in E. coli was successful, achieving rapid expression of a large amount of fructosyl transferase with catalyzing ability. This study could provide a theoretical basis for industrial production of fructosyl transferase.
